Genetically Encoded Photocontrol of Protein Localization in Mammalian Cells

Abstract

Precise photochemical control of protein function can be achieved through the site-specific introduction of caging groups. Chemical and enzymatic methods, including in vitro translation and chemical ligation, have been used to photocage proteins in vitro. These methods have been extended to allow the introduction of caged proteins into cells by permeabilization or microinjection, but cellular delivery remains challenging. Since lysine residues are key determinants for nuclear localization sequences, the target of key post-translational modifications (including ubiquitination, methylation, and acetylation), and key residues in many important enzyme active sites, we were interested in photocaging lysine to control protein localization, post-translational modification, and enzymatic activity. Photochemical control of these important functions mediated by lysine residues in proteins has not previously been demonstrated in living cells. Here we synthesized 1 and evolved a pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode the incorporation of this amino acid in response to an amber codon in mammalian cells. To exemplify the utility of this amino acid, we caged the nuclear localization sequences (NLSs) of nucleoplasmin and the tumor suppressor p53 in human cells, thus mislocalizing the proteins in the cytosol. We triggered protein nuclear import with a pulse of light, allowing us to directly quantify the kinetics of nuclear import.

Light-induced protein translocation by genetically encoded unnatural amino acid in Caenorhabditis elegans

“…In addition, we used a modified ONBK to increase the decaging efficiency which was reported previously (Fig. S3B) (Gautier et al, 2010). When modifi ed ONBK was added into liquid cultured stable transgenic strain, NLS TAG ::TagGFP was successfully read through and expressed.…”

“…ONBK is an unstable lysine derivative, and its ONB group is readily removed upon UV light exposure (Chen et al, 2009;Gautier et al, 2010). To test the functional consequence of ONBK incorporation, we employed two constructs as previously reported: 1) PCKRS (a PylRS mutant)/tRNA pair driven under the promoters of atp-2 and U6-10, respectively; 2) a nuclear localization signal with a lysine mutated to amber stop codon (shorted for NLS TAG ) conjuncted with TagGFP (a homologue of GFP) driven by the promoter atp-2 ( Fig.…”

How to obtain labeled proteins and what to do with them

“…Furthermore, the high concentrations of non-incorporated, free unnatural amino acid might be problematic, for example, if fluorescent amino acids are used for live cell imaging. At the moment, the most promising use of this method in cells therefore lies in applications where the free unnatural amino acid does not interfere with downstream applications, such as the photocaging of protein functions, as has recently been demonstrated by photocaging nuclear localization signals in proteins using a lysine bearing a photocleavable protecting group [71 ].…”

Rewiring translation – Genetic code expansion and its applications

“…Irradiating the cell with light of appropriate wavelength frees the side chain thereby activating the protein. Photo-caged versions of tyrosine (32), cysteine (33), serine (10) and lysine (34,35) have been genetically incorporated into proteins in bacteria, yeast and mammalian cells [54,75,[83][84][85][86]. As mentioned earlier, photocaged serine has been used to control the phosphorylation state of a serine residue and with it the localisation of a transcription factor in yeast [54].…”

“…As mentioned earlier, photocaged serine has been used to control the phosphorylation state of a serine residue and with it the localisation of a transcription factor in yeast [54]. The Chin lab demonstrated that photo-caged lysine can be used to control the recognition of nuclear localisation signals by importins in mammalian cells [85]. Light triggered activation of the NLS facilitated quantifying the kinetics of nuclear transport processes.…”